Activation of inflammatory pathways links obesity to insulin resistance. Inflammatory signaling pathways in obesity can be activated by excess nutrients (e.g. lipids). However, how excess nutrients are sensed within the cells and alter inflammatory program is fully understood. Two signaling proteins that may mediate this process are the two nutrient sensors: AMPK-activated protein kinase (AMPK) and SIRT. AMPK functions as a cellular energy gauge that regulates metabolic pathways in lipid and glucose metabolism. SIRT1, which also functions as an energy sensor, is an NAD+dependent deacetylase that mediates the effects of caloric restriction to extend longevity. AMPK and SIRT1 show striking similarities in sensing nutrient supply and regulating metabolic pathways and are likely to interact to perform these functions. Emerging evidence suggests that both AMPK and SIRT1 also regulate inflammatory signaling. Our preliminary data suggested that (1) AMPK and SIRT1 signaling and expression in macrophages and adipose tissue are down-regulated in conditions that evoke inflammation, such as exposure to lipopolysaccharide (LPS), free fatty acids (FFA), and diet-induced obesity;(2) activation of AMPK and SIRT1 prevents LPS- and FFA-induced inflammation in macrophages;(3) activation of AMPK stimulates SIRT1 activity and induces SIRT1 expression. Our overall hypothesis is that AMPK and SIRT1 serve as key determinants of inflammatory signaling events and AMPK and SIRT1 cooperate to regulate inflammation and insulin resistance in obesity. Specific Aim 1 will determine the protective effects of AMPK against FFA-induced inflammation in macrophages. We will examine the inflammatory signaling in LPS- and FFA-challenged macrophage with loss or gain of function of AMPK signaling. Specific Aim 2 will determine whether inactivation of AMPK in macrophages promotes inflammation and insulin resistance in diet-induced obese mice. We will perform bone marrow transplantation to generate mice with alpha1AMPK-deficient macrophages, and will characterize the inflammatory status and insulin sensitivity in these mice fed a high-fat diet or infused with lipids. Specific Aim 3 will determine whether SIRT1 mediates the protective effects of AMPK against FFA-induced inflammation in macrophages. We will examine the inflammatory signaling pathways in macrophages with loss or gain of function of SIRT1 paired with gain or loss of function of AMPK. We will also generate a mouse model deficient in SIRT1 in macrophages and assess inflammation and insulin sensitivity in these mice treated with a HF diet. This project will define AMPK and SIRT1 as negative regulators of lipid-induced inflammation and the novel roles of these proteins in bridging the signaling gap between nutrient metabolism and inflammation. The findings from this project will lead to the development of AMPK and SIRT1 as new therapeutic targets in treatment of obesity-induced inflammation and insulin resistance. PUBLIC HEALTH RELEVANCE: This goal of this project is to study the roles of AMPK and SIRT and their interaction in regulating inflammation and insulin resistance in obesity. The findings from this project will lead to the development of AMPK and SIRT1 as new therapeutic targets in treatment of obesity-induced inflammation and insulin resistance.